The way of Qu‐making significantly affected the volatile flavor compounds in Huangjiu (Chinese rice wine) during different brewing stages

Abstract The volatile flavor compounds of Huangjiu (Chinese rice wine) brewed from different raw materials were obviously different, but there were few studies on the volatile flavor compounds of Huangjiu brewed from different wheat Qu at different brewing stages. In this paper, headspace–solid phase microextraction combined with gas chromatography–mass spectrometry, combined with principal component analysis and sensory evaluation, was used to determine the volatile flavor compounds in Huangjiu brewed from wheat Qu made by hand and wheat Qu made by mechanical. The results showed that there were significant differences in the contents and types of volatile flavor substances in Huangjiu brewed from different wheat Qu at fermentation stages, and the prefermentation and postfermentation Huangjiu samples could be well distinguished from each other. Compared with the Huangjiu brewed from wheat Qu made by mechanical, the Huangjiu brewed from wheat Qu made by hand has stronger aroma and better taste.


| INTRODUC TI ON
Huangjiu (Chinese rice wine) is made from grain as the main raw material, wheat Qu or xiao Qu as saccharifying and fermenting agent (Ouyang et al., 2016), and is brewed from the processes of soaking rice, steamed rice, prefermentation, and postfermentation (Jiao et al., 2017). Huangjiu, with its unique flavor and high nutritional value (Ren et al., 2019;Zhao et al., 2018), is the national wine of modern China, and is known as one of the three ancient wines in the world. As a traditional Chinese alcoholic beverage (Gong et al., 2019;Yu et al., 2012), there are many varieties of Huangjiu, but due to the differences in various aspects such as wheat Qu and brewing technology, the final style of Huangjiu of different varieties is also different.
"Making wheat Qu with wheat and brewing wine with wheat Qu" is a traditional operation technology of Huangjiu brewing technology (GB/T13662-2018(GB/T13662- , 2018, the quality of wheat Qu can directly affect the quality and yield of Huangjiu (Shen, Yang, et al., 2012;Xu et al., 2010). Wheat Qu contains abundant microorganisms and enzymes , such as fungi, yeast, bacteria, saccharifying enzymes, and proteases (Plata et al., 2005), and these microorganisms play a very important role in the flavor of Huangjiu.
Nowadays, wheat Qu is divided into many types. According to different ways of making wheat Qu, wheat Qu can be divided into wheat Qu made by hand and wheat Qu made by mechanical. There are more or less differences in the flavor of Huangjiu brewed from these two kinds of wheat Qu. Thus, it is of great significance to study the volatile flavor compounds of Huangjiu brewed from different wheat Qu to control the flavor of Huangjiu in the industrial production process. Thus, it is of great significance to study the volatile flavor compounds of Huangjiu brewed from different wheat Qu to control the flavor of Huangjiu in the industrial production process.
With the development of economic globalization, Huangjiu has spread widely throughout the international market. Huangjiu is a complex mixture composed of hundreds of flavor compounds. The aroma of Huangjiu is one of the decisive factors affecting consumers' sensory quality and purchase (Shen, Zhu, et al., 2021;Welke et al., 2014). The aroma components of Huangjiu wine mainly come from raw materials, substances decomposed by various enzymes, and microbial metabolites in the brewing process (Chen & Xu, 2013;. At present, although there are many studies on the volatile flavor compounds of Huangjiu brewed with different raw materials, qualities, or technologies (Chen & Xu, 2012;Yong et al., 2013). However, there are few studies and experiments on the variation of volatile flavor compounds in different brewing stages of Huangjiu brewed from wheat Qu made by hand and wheat Qu made by mechanical. Therefore, it is necessary to study the changes of volatile flavor compounds in Huangjiu brewed from different wheat Qu during the different brewing stages.
In this study, HS-SPME-GC-MS (headspace-solid-phase microextraction combined with gas chromatography-mass spectrometry) was used to compare the composition changes of volatile flavor substances in Huangjiu brewed by wheat Qu made by hand and wheat Qu made by mechanical (Wang et al., 2014). The Huangjiu was brewed with the wheat Qu made by hand provided by Zhejiang Guyue Longshan Shaoxing Wine Co., Ltd. and the wheat Qu made by mechanical provided by Kuaijishan Shaoxing Wine Co., Ltd. The concentrations of ethanol, total acidity, and reducing sugar were determined. I hope this research can help provide more accurate critical control points for the production of better-flavored Huangjiu while preserving the flavor and superior quality characteristics of traditional Huangjiu, which will also have significance for the monitoring of Huangjiu production.

| Wheat Qu and chemicals
The Wheat Qu made by hand provided by Shaoxing Guyue Longshan Co., Ltd. and the Wheat Qu made by mechanical method provided by Kuaijishan Shaoxing Wine Co., Ltd. were used for brewing Huangjiu.
All reagents and standards were purchased at Sigma-Aldrich (Shanghai, China). Figure 1(a,b). The mechanical Wheat Qu process is basically the same as the manual Wheat Qu process, but the biggest difference is that the mechanical Wheat Qu process uses mechanical devices to replace part of the operating procedures in the manual Wheat Qu process ).

| Huangjiu making
There are two groups of Huangjiu samples in the experiment. One group is Huangjiu brewed from wheat Qu made by hand and the other group is Huangjiu brewed from wheat Qu made by mechanical method.

| Sample collection
In the process of fermentation, according to different times, samples

| Determination of chemical indices
The content of reducing sugar in rice wine samples was determined by the dinitrosalicylic acid method (Miller, 1959). Add 10 ml of Huangjiu sample into 50 ml of deionized water and determine the total acidity (in lactic acid) with 0 titration method 0.1 mol/L sodium hydroxide, until the end pH reaches 8.2 (GB/T13662-2018(GB/T13662- , 2018. Determination of ethanol content in Huangjiu is carried out by the distillation method (GB/T13662-2018, 2018).

| Volatile compound analysis
The SPME units and fibers (50 µm divinylbenzene (DVB)/carboxen (CAR)/polydimethylsiloxane (PDMS)) are from Supelco (Bellefonte, PA, USA). Each Huangjiu sample was diluted with deionized water to a final ethanol concentration of 6% (v/v) for accurate GC-MS analysis. In different stages, 2 g of sodium chloride was added into 5ml of sample and put into 20 ml headspace glass vials with screw caps, add 5 ml of internal standard (2-octanol, mass concentration 89.65 μg/L). The fibers were introduced into the SPME device, inserted into a headspace glass bottle, and shaken at 50°C for 30 min to extract and absorb volatile compounds. Then they were desorbed within 7 min at 250°C into the GC inlet with the automatic autosampler. A gas chromatograph (GC) with an attached mass spectrometer (SCION SQ-456; Bruker Daltonics Inc., USA) was used with a DB-Wax column (60 m*0.25 mm*0.25 μm; Agilent Technologies, USA). As the carrier gas, helium was delivered at a flow rate of 1 ml/ min. The gas chromatograph oven temperature was initially maintained at 40°C for 3 min, increased to 120°C at a rate of 6°C/min and then increased to 230°C at a rate of 8°C/min, and finally held for 15 min. The mass detector was operated in electron impact mode at an ionizing voltage of 70 eV. The ion source temperature was set at 220°C (Jung et al., 2014). The volatile components were identified by mass spectrometry (MS) and quantified by internal standard (2,4,6-trimethylpyridine, 1 mg/L). The relative content of each component was counted according to the area normalization method.
The results were reported in the mean value of three replicates of Huangjiu samples (Yang et al., 2017).

| DNA extraction and sequencing
DNA extraction and sequencing were performed according to Xie, GF method . A linear relationship was established between the sequencing data and flavor substances by partial least squares regression. The X variable is relative abundance of bacterial community. The Y variable is the average concentration of flavor substances (Shen, Zhu, et al., 2021;.

| Sensory analysis
Sensory evaluation of the Huangjiu was conducted by a panel of 10 judges (5 males and 5 females) with rich experience in wine descriptive analysis. There was a uniform source of lightening, absence of noise, and distracting stimuli. Fifteen milliliter samples were and covered with petri dishes. A total of 11 descriptors of appearance (color and turbidity), aroma (alcohol, fruit, and cereal), taste/ flavor (sweet, sour, and bitter), and mouthfeel (astringency, continuation, and full body) were selected by the panel during preliminary training sessions to describe the samples. Standards used to define the aromas and taste descriptor were included in the training and wine-tasting test. Intensity ratings were scored on a scale from 0 to 9 (0: none; 1-2: very weak; 3-4: ordinary; 5-6: moderate; 7-8: strong; 9: very strong).

| Statistical analysis
The chemical composition and flavor of each rice wine were analyzed, and the analysis was repeated three times, and the results were expressed as the average value ± standard deviation. SPSS ver.

| Analysis of general chemical indicators during fermentation
The contents of ethanol, total acidity, and reducing sugar in Huangjiu samples are shown in Table 1. The content of ethanol and reducing sugar in Huangjiu can reflect the liquefaction and saccharification of wheat Qu, thus reflecting the fermentation performance of wheat Qu. Alcohol is the carrier of aroma and flavor substances in Huangjiu, which can make Huangjiu have the mellow aroma. The experimental results showed that the alcohol content of Huangjiu brewed from wheat Qu made by hand was 15.61% vol, which was significantly lower than that of Huangjiu brewed from wheat Qu made by mechanical was 17.13% vol; correspondingly, the reducing sugar content of Huangjiu brewed from wheat Qu made by hand was 4.01 g/L, which was significantly higher than that of Huangjiu brewed from wheat Qu made by mechanical which was 1.13 g/L. This is because in the process of Huangjiu fermentation, the reducing sugar in the mash is converted to ethanol through the glycolytic pathway (Embden-Meyerhof-Parnas (EMP)) by the action of yeast (Zhang, Wang, et al., 2019). This indicates that the liquefaction and saccharification forces of wheat Qu made by mechanical are obviously stronger than those of wheat Qu made by hand.
Acidity is an important index to judge the fermentation degree of Huangjiu. "No acid, no taste" indicates that acidities are also an important part of flavor compounds in Huangjiu. Proper amount of acid can not only enhance the taste of Huangjiu, but can also make the color and aroma of Huangjiu more harmonious and plump (Yu et al., 2012). Therefore, acidity detection is an essential management method during Huangjiu fermentation. By analyzing the acid content of Huangjiu, it is helpful to know the fermentation situation of Huangjiu in time and ensure the normal fermentation process. Table 1 shows that the acidity of Huangjiu brewed from wheat Qu made by hand was 3.16 g/L, which is lower than the acidity of the Huangjiu brewed from wheat Qu made by mechanical which was 3.53 g/L. Compared with the Huangjiu brewed from wheat Qu made by hand, the Huangjiu brewed from wheat Qu made by mechanical had higher total acidity and lower residual sugar, which indicated that the Huangjiu brewed from wheat Qu made by mechanical had excellent fermentation performance. In our previous study, we found that the abundance of lactic acid bacteria in the two kinds of wine is different due to the different microorganisms brought in by different wheat Qu, which is one of the main reasons for the inconsistent acidity of rice wine.

| Volatile flavor compounds in the different fermentation stages of Huangjiu making
Qualitative analysis of the main aroma compounds in the Huangjiu made from wheat Qu by hand and in the Huangjiu made from wheat Qu by mechanical method during manufacture is presented in Tables 2 and 3. Sixty major flavor compounds were identified, which included 18 alcohols, 26 esters, 9 aldehydes, and 7 acids.
During the 2, 4, and 6 days of the primary fermentation, 60 vol-

TA B L E 3
The contents of volatile flavor compounds during different brewing stages of Huangjiu fermented with the Huangjiu made from wheat Qu by mechanical method which were analyzed by headspace-solid phase microextraction combined with gas chromatography-mass spectrometry (HS-SPME-GC-MS)

TA B L E 3 (Continued)
content of components, particularly alcohol and esters, during the fermentation process of Huangjiu.

| Changes in the main volatile aroma compounds during different brewing stages
The flavor of Huangjiu does not exist alone, but is formed by the interaction of alcohols, esters, and aldehydes (Cao et al., 2012;Xu et al., 2015). In terms of the types of volatile compounds, Huangjiu  The key substances detected in alcohols include ethanol, isobutanol, β-phenylethanol, and isopentanol, which account for much of the total content of alcohols. At the third time point of postfermentation, the content of ethanol in Huangjiu brewed from wheat Qu made by hand was lower than that of Huangjiu brewed from wheat Qu made by mechanical, but the difference was not significant. The content of isobutanol in the Huangjiu brewed from wheat Qu made by hand is higher than that of the Huangjiu brewed from wheat Qu made by mechanical. The content of isopentanol in the Huangjiu brewed from wheat Qu made by hand is lower than that in the Huangjiu brewed from wheat Qu made by mechanical. The content of β-phenylethanol in the Huangjiu brewed from wheat Qu made by hand is lower than that in the Huangjiu brewed from wheat Qu made by mechanical. The total amount of alcohols in the Huangjiu brewed from wheat Qu made by hand is significantly higher than that in the Huangjiu brewed from wheat Qu made by mechanical. This may be because the content of amino acids in wheat Qu made by hand is higher than that of wheat Qu made by mechanical. During the brewing process of Huangjiu, the amino acids contained in Huangjiu are successively absorbed by yeast and then converted into higher alcohols by the Ehrlich pathway (Hazelwood et al., 2008).

| Flavor assay based on the ratio of higher alcohols to esters
The aroma of Huangjiu is composed of many kinds of flavor compounds, among which the main aroma compounds are alcohols, esters, and aldehydes (Zhang et al., 2013). Advanced alcohol-to-ester ratio, which is a reliable index for evaluating the flavor characteristics of distillate spirits (Landaud et al., 2012;Procopio et al., 2011), was used to assess flavor in the Huangjiu brewed in this research. The fragrance of esters was more highlighted when the proportion was 2 and was thought to be moderate at a proportion between 3 and 4. If the ratio exceeded 5, the alcohol aroma would be highlighted.
Advanced alcohol-to-ester ratio of the two Huangjiu (Figure 4) remained almost stable during the stages of postfermentation stage but apparently fluctuated by primary fermentation stage, ranging from 6 to 4. In the fermentation process, advanced alcohol-to-ester ratio of the two kinds of wine was kept at about 3, and the aroma of Huangjiu made by hand is superior to that made by mechanical method.
F I G U R E 7 Sensory profiles of organoleptic attributes of Huangjiu made with different ways to make wheat Qu

| Sensory evaluation
Sensory analysis of the Huangjiu made by the different ways to make wheat Qu was performed by evaluating the organoleptic quality. The intensity ratings of the Huangjiu made from wheat Qu by hand and the Huangjiu made from wheat Qu by mechanical method are presented in (Figure 7). As for appearance, the Huangjiu made from wheat Qu by hand exhibited a lower turbidity score (5.7) among the two Huangjiu, while the two wines exhibited a similarly strong intensity rating in yellowness. The sweet flavor in the two Huangjiu seemed to have a lower descriptor score (<4.0) than the other aroma and taste. The two wines had big different ordinary values of fruit-aroma attribute, which may have resulted from the different content esters produced through fermentation (Erten et al., 2007). Compared with the Huangjiu made from wheat Qu by mechanical method, the Huangjiu made from wheat Qu by hand has a little strong alcoholic aroma. The attribute levels of bitter flavor, astringency, and cereal-aroma were generally similar between two wines, even though the Huangjiu made from wheat Qu by mechanical method showed a higher intensity of sour flavor. The Huangjiu brewed from wheat Qu by hand has been highly assessed in continuation and full-body mouthfeel.

| CON CLUS ION
The volatile flavor compounds and chemical analysis of Huangjiu made by the wheat Qu made by hand and the wheat Qu made by mechanical method were investigated. Headspace-solid phase microextraction coupled with gas chromatography-mass spectrometry showed that wine brewed by different methods of making wheat Qu differed mainly in the numbers and amounts of alcohols and esters. In general, with the promotion of a fermentation process, the volatile components of handmade wheat Qu Huangjiu were always taller than that of the mechanical production of Huangjiu, which may be due to the differences in production technology between hand and mechanical wheat Qu. Wheat Qu made by mechanical method uses machine pressing, while wheat Qu made by manual method uses manual trampling. The pressure of the machine is often greater than the pressure of manual trampling, so the gap of wheat Qu made by mechanical method is smaller, the air content is less, and mold and yeast will not grow well. Moreover, the moisture content of mechanical wheat Qu is lower than that of manual wheat Qu, which also affects the growth and reproduction of microorganisms in the Qu to a certain extent. Therefore, the difference in composition of Huangjiu with different Qu-making methods may be mainly on account of the difference in microorganism species in wheat Qu. The effect of microorganisms on the volatile flavor compounds of Huangjiu needs further study.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author, upon request.